We want to analyze chemo-mechanical coupling in muscle. We will use coordinated X-ray diffraction and electron microscopy to study the behavior of cross-bridges and myofilaments in striated muscle. Our X-ray diffraction experiments should proceed with unprecedented speed, because we will build and employ a powerful new X-ray detector which promises to replace X-ray film in most experiments since it is noise-free and is about 100 times faster. Our electron microscopy of myofibrillar structure should develop toward unprecedented freedom from artifacts because we will monitor the X-ray diagram of muscle during specimen preparation, and will systematically vary preparation techniques so as to stabilize the diffraction pattern throughout the fixation, dehydration and embedding. We will use glycerinated muscle fibers, so that myofibrils can be directly affected by ATP, its analogues (e.g., AMPOPNP, AMPCPOP), various protein-modifying agents (e.g., NEM, PCMB), and solution additives (glycols), and so that chemical fixatives (glutaraldehyde, TAPO, trinitro aromatics) can just as directly fix myofibrils without first affecting membrane-mediated regulation of contractile behavior. We will emphasize insect flight muscle (Lethocerus) because it provides the best EM images of cross-bridges, the best-understood relationship between X-ray patterns and different cross-bridge lattice patterns (rigor vs. relaxed), but vertebrate striated muscle, including glycerinated or fixed autopsy and clinical biopsy material, should become feasible later in the project. Many fibers will be mounted in a thermostatted bath on a transducer and observed by phase contrast or polarization microscopy, to permit temperature manipulation and tension recording, as well as videotaped records of sarcomere patterns and birefringence during exposure to chemicals and fixatives, so that we may determine function before we determine structure. Optical and computer image processing will be employed to make the most of good EMs of the cross-bridges.